JP2000060575A - Wnt-6 polypeptide and wnt-6 polynucleotide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new Wn t-6 polypeptide for treatment, diagnosis or the like for neuropathy, e.g. Alzheimer disease or the like, cancer, cardiovascular disease, attack, developmental disability or the like which contains the amino acid sequence having high homology over the whole length to a specific amino acid sequence. SOLUTION: This peptide is an isolated new Wn t-6 polypeptide containing the amino acid sequence having at least 98% of homology over the whole length of the formula to the amino acid sequence of the formula and the Wn t-6 polypeptide and Wn t-6 polynucleotide are useful in the treatment, the diagnostic assay or the like for functional disorders or diseases containing neuropathy (particularly schizophrenia, bipolar disorder, unipolar disorder, Alzheimer disease or epilepsia), cancer (particularly squamous cell carcinoma), cardiovascular disease, attack and developmental disability (lamellar ichthyosis is contained). Another objective polypeptide is obtained by integrating polynucleotides obtained by standard cloning and screening from the cDNA library arising from the human heart by the use of EST analysis into the expression system to express in the host cell.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a newly identified polypeptide, a polynucleotide encoding the polypeptide, an agonist, an antagonist which may be effective in or in the treatment of the polypeptide and the polynucleotide. And / or use in identifying compounds that are inhibitors and methods of producing the polypeptides and polynucleotides.

[0002]

2. Description of the Prior Art The drug discovery process is currently undergoing radical changes. Because it extends to "functional genomics," that is, high-throughput genomic or gene-based biology. This approach as a means to identify genes and gene products as therapeutic targets is rapidly replacing relatively early approaches based on "positional cloning." Phenotype,
That is, the biological function or genetic disease will be identified, and then the pathogenic gene will be located by the location of its genetic map. Functional genetics science relies heavily on high-throughput DNA sequencing technology and various tools in bioinformatics to identify gene sequences of interest from many of the molecular biology databases currently available. ing. There is still a need to identify and characterize as yet undetermined genes and their related polypeptides / proteins as targets for drug discovery.

[0003]

The present invention relates to Wnt-6, in particular Wnt-6 polypeptides and Wnt-6 polynucleotides, recombinant materials and methods for their production. In another aspect, the invention provides a neurological disorder (especially schizophrenia, bipolar disorder, unipolar disorder, Alzheimer's disease and epilepsy), cancer (especially squamous cell carcinoma), cardiovascular disease, stroke, and development. The present invention relates to a method for using the above-mentioned polypeptide and polynucleotide, including the treatment of disorders (including ichthyosis vulgaris) (hereinafter, collectively referred to as “the disease”). In another aspect, the invention provides a method of identifying agonists and antagonists / inhibitors using the agents provided by the invention, and treating the conditions associated with Wnt-6 imbalance using the identified compounds. Regarding In yet another embodiment, the invention provides inappropriate Wnt-6 activity or W
It relates to diagnostic assays for detecting diseases associated with nt-6 levels.

[0004]

SUMMARY OF THE INVENTION In one aspect, the present invention relates to Wnt-6 polypeptides. Peptides of this type include an isolated poly-peptide comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2, preferably at least 99%. Peptides are included. One such polypeptide includes the amino acid sequence of SEQ ID NO: 2.

In another peptide of the invention, the isolated amino acid sequence has at least 98% identity, preferably at least 99% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. Included polypeptides. Such a polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2. Further peptides of the invention include an isolated polypeptide encoded by a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1.

The polypeptides of the present invention are considered to be members of the Frizzled receptor family. Therefore, I am interested in them. Because this protein is
It functions as a transduction molecule for developmental processes during Wnt signaling. This is essential for normal morphogenesis and / or differentiation function in diverse tissues. The involvement of the Wnt signaling cascade in neuropathy has been established in several studies. di
Mice knocked out of shevelled-1 (downstream transducer of Wnt-1 type signaling) show a phenotype with overt psychological and social interaction abnormalities, which is associated with human schizophrenia. Closely related to the disease (Lijam et al., 1997 Cell 90: 895-905). More recently, Cotter et al. (Neuroreport 9: 1379-1383, 1998).
Is based on the significant difference in β and γ-catenin distribution in healthy and schizophrenic patients, and
Reported an abnormality in t signaling. Wnt-6 is not transformed and does not induce cell axis duplication Wn-6
t belongs to the class of ligands. Wnt ligands are thought to transduce signals that block the signals of the transforming class of Wnts, including Wnt-1, Wnt-3a and Wnt-8, which in turn act to suppress disheveled. Thus, aberrant Wnt-6 activity may induce a phenotype similar to the dishelled knockout phenotype. Thus, Wnt-6 is a strong candidate for schizophrenia and related neuropathy (eg, bipolar disease). Small molecule agonist / antagonists or antibodies and antisense sequences corresponding to Wnt-6 can be used as a medicament for the treatment of the above disorders and various other disorders. These properties are hereinafter referred to as "Wnt-6 activity" or "Wnt-6 polypeptide activity" or "biological activity of Wnt-6". Among these activities are also the antigenic and immunogenic activities of said Wnt-6 polypeptides, in particular the antigenic and immunogenic activities of the polypeptide of SEQ ID NO: 2. The polypeptides of the present invention preferably exhibit at least one biological activity of Wnt-6.

The polypeptide of the present invention may be in the form of a "mature" protein or may be part of a larger protein, such as a precursor or fusion protein. Often it is advantageous to include additional amino acid sequences,
Such amino acid sequences may include secretory or leader sequences, prosequences, sequences that aid purification, such as multiple histidine residues, or additional sequences that ensure stability during recombinant production.

The present invention also includes a variant of the above-mentioned polypeptide, that is, a polypeptide which differs from the reference polypeptide by conservative amino acid substitution (one residue is replaced by another residue having similar properties). included. Typical such substitutions are between Ala, Val, Leu and Ile; Ser and Thr.
Between; acidic residues Asp and Glu; between Asn and Gln; basic residues Lys and Arg; or aromatic residues Phe and Tyr
Happens in between. In particular, several, 5-10, 1-5, 1
Variants in which ~ 3, 1-2 or 1 amino acids are substituted, deleted or added in any combination are preferred.

The polypeptide of the present invention can be produced by any appropriate method. Such polypeptides include isolated natural polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides,
Alternatively, a polypeptide produced by a combination of these methods is included. Means for producing such polypeptides are well understood in the art.

In a further aspect of the invention, the invention comprises
Wnt-6 polynucleotide. Such polynucleotides include isolated polynucleotides that comprise a nucleotide sequence that encodes a polypeptide having at least 98% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. Be done. In this regard, polypeptides having at least 99% identity are preferred. Examples of such a polynucleotide include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 which encodes the polypeptide of SEQ ID NO: 2.

A further polynucleotide of the invention is at least 9 nucleotides over the entire coding region for the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2.
Included is an isolated polynucleotide comprising a nucleotide sequence having 0% identity, preferably at least 95% identity. In this regard, at least 97
Polynucleotides with% identity are more preferred, those with at least 98-99% identity are even more preferred, and those with at least 99% identity are most preferred.

A further polynucleotide of the present invention comprises a nucleotide sequence having at least 90% identity, preferably at least 95% identity to the polynucleotide of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. Including isolated polynucleotides. In this regard, polynucleotides having at least 97% identity are more preferred, but those with at least 98-99% identity are even more preferred, and at least 99%.
Most preferred are polynucleotides having the same identity. Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 and the polynucleotide of SEQ ID NO: 1. The invention also provides polynucleotides which are complementary to all the above polynucleotides.

The nucleotide sequence of SEQ ID NO: 1 is Wnt-6-mo
Shows homology with use (Gavin et al., 1990 Gene Dev. 4, 2319-2332). The nucleotide sequence of SEQ ID NO: 1 is a cDNA sequence and includes a polypeptide coding sequence (nucleotides 224-1321) that encodes a polypeptide of 365 amino acids that is the polypeptide of SEQ ID NO: 2. Even if the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 is the same as the polypeptide coding sequence contained in SEQ ID NO: 1, due to the redundancy (degeneracy) of the genetic code, the polypeptide of SEQ ID NO: 2 is still used. It may be a sequence other than the sequence encoded and included in SEQ ID NO: 1. The polypeptide of SEQ ID NO: 2 is structurally related to other proteins of the Frizzled receptor family and is Wnt-6-mouse (Gavin et al., 1990.
Genes Dev. 4, 2319-2332) with homology and / or structural similarity.

The preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions / properties to the homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention include at least one Wnt-.
Has 6 activities.

The polynucleotides of the invention were analyzed by EST analysis from a cDNA library derived from mRNA in human heart cells by standard cloning and screening (Adams, MD et al., Science (1991) 252:
1651-1656; Adams, MD et al., Nature (1992) 355: 632-63.
4; Adams, MD et al., Nature (1995) 377 Supp: 3-174). Further, the polynucleotide of the present invention can be obtained from a natural source such as a genomic DNA library, or can be synthesized by using a known technique which is commercially available.

When the polynucleotide of the present invention is used for recombinant production of the polypeptide of the present invention, the polynucleotide may include the coding sequence of the mature polypeptide alone or other coding sequences (eg, leader or secretion). Sequence, pre-, pro- or pre-pro-protein sequence, or other fusion peptide portion) in the same reading frame as the coding sequence for the mature polypeptide. For example, a marker sequence that facilitates purification of the fusion polypeptide can be encoded.
In a preferred embodiment of this aspect of the invention, the marker sequence is provided by the pQE vector (Qiagen, Inc.) and is provided by Gentz et al., Proc. Natl. Acad. Sci. USA (1989) 86: 8.
21-824, a hexa-histidine peptide, or HA tag. The polynucleotide may also include 5'and 3'non-coding sequences, such as transcribed but untranslated sequences, splicing and polyadenylation signals, ribosome binding sites, and mRNA stabilizing sequences.

As a further embodiment of the present invention, several amino acid residues, for example 5 to 10, 1 to 5, 1 to 3, 1 to 2, or 1 amino acid residue may be substituted in any combination. , A polynucleotide encoding a polypeptide variant comprising the amino acid sequence of SEQ ID NO: 2 which has been deleted or added.

A polynucleotide which is identical or sufficiently identical to the nucleotide sequence contained in SEQ ID NO: 1 has been used for the isolation of full-length cDNA and genomic clones encoding the polypeptide of the invention and also in SEQ ID NO: 1. CDNAs and genomes of other genes having a high sequence similarity to (including human-derived paralogs and genes encoding orthologs and paralogs derived from species other than humans) To isolate clones, as hybridization probes for cDNA and genomic DNA, or for nucleic acid amplification (P
It can be used as a primer for CR) reaction.
Generally, these nucleotide sequences are 70%, preferably 80%, more preferably 90%, most preferably 95% identical to a reference nucleotide sequence. The probe or primer usually contains 15 or more nucleotides, preferably 30 or more nucleotides and may have 50 or more nucleotides. Particularly preferred probes are those having a range of 30-50 nucleotides. Particularly preferred primers are those having a range of 20-25 nucleotides.

The polynucleotide encoding the polypeptide of the present invention (including homologues derived from species other than human) is subjected to stringent hybridization using a labeled probe having the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof. Screening a suitable library under conditions, full-length cDNA containing the polynucleotide sequence
A and a genomic clone are obtained by a method comprising steps. Such hybridization techniques are known to those of skill in the art. Preferred stringent hybridization conditions are 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate).
, 50 mM sodium phosphate, pH 7.6, 5x Denhardt's solution, 10% dextran sulfate and 20 µg / ml denatured and sheared salmon sperm DNA were incubated overnight at 42 ° C, then the filters were washed with 0.1x SSC. About 65
Including washing at ℃. Thus, the invention also includes a polynucleotide obtained by screening a suitable library under stringent hybridization conditions with a labeled probe having the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof.

As will be appreciated by those in the art, in many cases the region encoding the polypeptide will be 5'of the cDNA.
Due to the short ends, the isolated cDNA sequence will be incomplete. It is due to reverse transcriptase, which originally has a low "processivity" (processivity: a measure of the enzyme's ability to remain bound to the template during the polymerization reaction), which causes mRNA template during first strand cDNA synthesis. Can not complete the DNA copy of.

To obtain full-length cDNA, or short chain c
There are several methods known and available to those of skill in the art for extending DNA, such as those based on cDNA terminal rapid amplification (RACE) (eg, Frohman).
Et al., PNAS USA 85, 8998-9002, 1988). Recent improvements in the above techniques, such as those demonstrated by Marathon ™ technology (Clontech Laboratories Inc.), have greatly facilitated the search for longer cDNAs. Marathon
In the TM technique, cDNA is prepared from mRNA extracted from a given tissue and an "adapter" sequence is ligated to each end. Subsequently, nucleic acid amplification (PCR) is performed using a combination of gene-specific and adapter-specific oligonucleotide primers to amplify the "deletion"5'end of the cDNA. Next, a "nested" primer, ie, a primer designed to anneal within the amplification product (typically an adapter-specific primer that anneals further 3'to the adapter sequence and an additional 5'to the known gene sequence). The PCR reaction is repeated with a gene-specific primer that anneals to the side. Then, the product of this reaction was analyzed by DNA sequencing, and the product was analyzed for existing c
Full-length cDNA can be constructed either by direct binding to DNA or by performing another full-length PCR using new sequence information for 5'primer design.

The recombinant polypeptide of the present invention can be produced from a genetically engineered host cell containing an expression system using methods known in the art. Thus, in a further aspect, the invention relates to expression systems containing one or more polynucleotides of the invention, host cells genetically engineered by the expression systems, and the production of polypeptides of the invention by recombinant methods. Cell-free translation systems can also be used to produce proteins of this type using RNA derived from the DNA constructs of the invention.

For recombinant production, host cells are genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Introduction of polynucleotides into host cells is described by Davis et al., Basic Methods in Molecular Bi
ology (1986) and Sambrook et al., Molecular Cloning:
A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY (1989)
Can be done by the methods described in many standard laboratory manuals such as Suitable such methods include, for example, calcium phosphate transfection,
DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic introduct
ion) or infection.

As typical examples of suitable hosts, bacterial cells (eg Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (eg yeast, Aspergillus), insect cells (eg Drosophila S.
2, Spodoptera Sf9), animal cells (eg CHO, C)
OS, HeLa, C 127, 3T3, BHK, HEK 29
3, Bowes melanoma cells) and plant cells.

A wide variety of expression systems can be used. Such expression systems include, among others, chromosomal, episomal and viral derived systems such as bacterial plasmid derived, bacteriophage derived, transposon derived, yeast episome derived, insert factor derived, yeast chromosomal element derived, virus (eg baculovirus, SV40 Such as papovavirus, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, retrovirus), and vectors derived from combinations thereof, for example, plasmids such as cosmids or phagemids and genetics of bacteriophages. Some come from elements. These expression systems may include control sequences that regulate as well as direct expression.
In general, any system or vector capable of maintaining, amplifying and expressing a polynucleotide for production of a polypeptide in a host may be used. Sambrook et al, Mo
The appropriate nucleotide sequence may be inserted into the expression system by any of the well-known and routine techniques, such as those described in lecular Cloning: A Laboratory Manual (supra). Appropriate secretion signals may be incorporated into the polypeptide of interest in order to secrete the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular milieu. These signals may be endogenous or heterologous to the polypeptide of interest.

If the polypeptide of the invention is to be expressed for use in screening assays, it is generally preferable to produce the polypeptide on the surface of cells. In that case, cells are harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium is harvested to recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed and then the polypeptide recovered.

For recovering and purifying the polypeptide of the present invention from recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity is used. Known methods including chromatography, hydroxylapatite chromatography and lectin chromatography can be used. Most preferably,
High performance liquid chromatography is used for purification. When the polypeptide is denatured during intracellular synthesis, isolation and / or purification, known techniques for refolding proteins can be used to restore the active conformation.

The present invention also relates to the use of the polynucleotide of the present invention as a diagnostic agent. Associated with dysfunction,
Detection of a mutant form of the gene characterized by the polynucleotide of SEQ ID NO: 1 is useful for diagnosing a disease caused by underexpression, overexpression, or spatially or temporally altered expression of the gene or susceptibility to the disease. It would provide a diagnostic tool that could be added or made its diagnosis. Individuals with mutations in the gene can be found at the DNA level by various techniques.

The nucleic acid for diagnosis can be obtained from cells of a subject, for example, cells derived from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection, or it may be enzymatically amplified using PCR or other amplification methods prior to analysis. RNA or cDNA can also be used in a similar manner.
Deletion and insertion mutations can be detected by a change in the size of the amplification product as compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled Wnt-6 nucleotide sequences. Perfectly matched sequences and mismatched duplexes can be distinguished by RNase digestion or by differences in melting temperature. Also, D
NA sequence differences can also be detected by alterations in the electrophoretic mobility of DNA fragments on gels with or without denaturing agents, or by direct DNA sequencing (eg, Myers et al., Science (1985) 230: See 1242). Sequence changes at specific positions can also be confirmed by nuclease protection assays (eg, RNase and S1 protection) or chemical cleavage methods (Co
tton et al., Proc. Natl. Acad. Sci. USA (1985) 85: 4397-
See 4401). In another embodiment, Wnt-6 is used, for example, for efficient screening of gene mutations.
An array of oligonucleotide probes containing the nucleotide sequence or fragments thereof can be constructed. Array techniques are well known and have general applicability,
It has been used to solve various problems in molecular genetics, including gene expression, genetic linkage and genetic variability (eg, M. Chee et al., Science, Vol.274, pp.61).
0-613 (1996)).

The diagnostic assay is Wnt-6 according to the method described above.
A method for diagnosing or determining susceptibility to the disease is provided by detecting a mutation in a gene. In addition, a polypeptide or mRN from a sample obtained from the subject
A method of measuring an abnormal decrease or increase in the level of A can make a diagnosis of said disease. The decrease or increase in the expression can be achieved by a polynucleotide quantification method known in the art, such as nucleic acid amplification (eg, PCR, RT-PCR), R
RN by either Nase protection, Northern blotting or other hybridization method
It can be measured at the A level. Assay techniques for measuring the levels of proteins such as the polypeptides of the invention in a sample obtained from a host are well known to those of skill in the art. Such assay methods include radioimmunoassay, competitive binding assay, Western blot analysis, EL
ISA assay etc.

Thus, in another embodiment, the invention provides: (a) a polynucleotide of the invention (preferably the nucleotide sequence of SEQ ID NO: 1) or a fragment thereof, (b)
a nucleotide sequence complementary to the nucleotide sequence of (a),
(c) A polypeptide of the present invention (preferably the polypeptide of SEQ ID NO: 2) or a fragment thereof, or (d) an antibody against the polypeptide of the present invention (preferably the polypeptide of SEQ ID NO: 2). Diagnostic kit. It will be appreciated that in such a kit, (a), (b), (c) or (d) is a substantial constituent.
Such a kit may be a disease or a susceptibility to a disease, especially a neurological disorder (especially schizophrenia, bipolar disorder, unipolar disorder, Alzheimer's disease and epilepsy), cancer (especially squamous cell carcinoma), cardiovascular disease, stroke. , And is useful for diagnosing developmental disorders (including ichthyosis).

The nucleotide sequence of the present invention is also useful for chromosome identification. This sequence can target and hybridize to a particular location on an individual human chromosome. Generating a chromosomal map of related sequences according to the present invention is an important first step in correlating these sequences with gene-related disorders. Once a sequence is mapped to a precise chromosomal location, the physical location of that sequence on that chromosome can be correlated with genetic map data. This type of data is, for example, V. McKusick, Mendelian Inheritance in Man (J
Found online at ohns Hopkins University Welch Medical Library). Then, the relationship between the genes mapped to the same chromosomal region and the disease is confirmed by linkage analysis (co-inheritance of physically adjacent genes).

Differences in the cDNA or genomic sequence between affected and unaffected individuals can also be examined. If a mutation is observed in some or all of the affected individuals, but not in any normal individual, then the mutation may be responsible for the disease. The gene of the present invention is human chromosome 2q34-q.
Mapped to 36.

The nucleotide sequences of the present invention are also useful for tissue localization. By such a technique, the expression pattern of human Wnt-6 polypeptide in tissues can be
It can be determined by detecting mRNAs encoding these. These techniques include in situ hybridization techniques, and nucleotide amplification techniques (eg PCR). Such techniques are well known in the art. The results of these studies provide an indication of the normal function of the polypeptide in the organism. Furthermore, the normal expression pattern of human Wnt-6 mRNA and human Wn-6
By comparison with the expression pattern of the mRNA encoded by the t-6 gene, the role of mutant human Wnt-6 polypeptides in disease states or normal human Wnt-6
A valuable insight into the role of inappropriate expression of polypeptides is obtained. Such inappropriate expression may be temporal, spatial or simply quantitative in nature.

The polypeptides of the present invention, fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for the polypeptides of the present invention. By "immunospecific" is meant that the antibody exhibits a substantially higher affinity for a polypeptide of the invention than its affinity for other related polypeptides in the prior art.

An antibody against the polypeptide of the present invention can be obtained by administering a fragment, analog or cell containing the polypeptide or epitope to an animal (preferably a non-human animal) using a conventional protocol. For preparation of monoclonal antibodies, any technique which produces antibodies from continuous cell line cultures can be used. For example, hybridoma method (Kohler, G. and Milstein, C., Nature (1975) 256: 495-497), trioma method, human B cell hybridoma method (Kozbor et al., Immun).
ology Today (1983) 4:72) and the EBV-hybridoma method (Cole et al., Monoclonal Antibodies and Cancer T.
herapy, pp.77-96, Alan R. Liss, Inc., 1985).

To prepare single chain antibodies to the polypeptides of the present invention, the methods for preparing single chain antibodies as described in US Pat. No. 4,946,778 can be adapted.
Also, transgenic mice, or other organisms including other mammals, may be utilized to express humanized antibodies. Using the above antibody, a clone expressing the polypeptide can be isolated and identified, or the polypeptide can be purified by affinity chromatography. Antibodies against the polypeptide of the present invention,
In particular, it may be used for the treatment of the abovementioned diseases.

In a further aspect of the invention, the invention comprises:
A genetically engineered preparation comprising the polypeptide of the present invention or a fragment thereof and various portions of constant regions of H chain or L chain of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE) Related to soluble fusion proteins. As an immunoglobulin, human IgG, especially I
The constant region of the heavy chain of gG1 is preferred, in which case the fusion occurs in the hinge region. In a particular example, the Fc portion can be easily removed by incorporating a cleavage sequence that can be cleaved by blood coagulation factor Xa. Furthermore, the invention relates to methods of genetically engineering these fusion proteins and their use in drug screening, diagnosis and therapy. Also, a further aspect of the present invention relates to a polynucleotide encoding such a fusion protein. Examples of fusion protein technology are international patent applications WO94 / 29458 and WO94 / 22914
Can be found in.

A further aspect of the present invention relates to a method of eliciting an immunological response in a mammal, the method being sufficient to generate an antibody and / or T cell immune response, especially to protect said animal from said disease. Comprising inoculating a mammal with a polypeptide of the invention. Yet another aspect of the invention is to elicit an immunological response to produce antibodies that protect the mammal from the disease in.
A method of eliciting an immunological response in a mammal comprising delivering the polypeptide in vivo via a vector that directs the expression of a polynucleotide encoding the polypeptide of the invention.

A further aspect of the present invention relates to an immunological / vaccine formulation (composition) which, when introduced into a mammalian host, elicits an immunological response against the polypeptide of the present invention in the mammal. Contains a polypeptide or polynucleotide of the invention. The vaccine formulation may further include a suitable carrier. Since the polypeptide may be broken down in the stomach, it is preferably administered parenterally (eg by subcutaneous, intramuscular, intravenous or intradermal injection). Formulations suitable for parenteral administration include sterile aqueous and non-aqueous injection solutions that may contain antioxidants, buffers, bacteriostats and solutes that make this formulation isotonic with the blood of the recipient, and suspensions. There are aqueous and non-aqueous sterile suspensions which may include agents or thickeners. Such formulations may be presented in single-dose or multi-dose containers (eg, sealed ampoules and vials) and may be stored in lyophilized form with the addition of a sterile liquid carrier just prior to use. it can. The vaccine formulation may include an adjuvant system to enhance the immunogenicity of the formulation, such as an oil-in-water adjuvant system or any other adjuvant system known in the art. The dose varies depending on the specific activity of the vaccine, but can be easily determined by routine experimentation.

The polypeptides of the present invention are involved in one or more biological functions, including one or more pathological conditions, especially the diseases mentioned above. Therefore, it is desirable to develop screening methods to identify compounds that stimulate or inhibit the function of this polypeptide. Accordingly, in a further aspect, the invention provides a method of screening compounds to identify compounds that stimulate or inhibit the function of this polypeptide. Generally, agonists or antagonists are used for the therapeutic and prophylactic purposes of said diseases. Compounds can be identified from a variety of sources, such as cells, cell-free preparations, chemical libraries and mixtures of natural products. The agonist, antagonist or inhibitor thus identified may optionally be a natural or modified substrate, ligand, receptor, enzyme, etc. of the polypeptide, and
It may be a structural or functional mimetic (Coligan et al., Current Protocols in Immunology 1 (2):
See Chapter 5 (1991)).

In the screening method, the binding of the candidate compound to the polypeptide of the present invention, the cell or membrane carrying the polypeptide, or its fusion protein is carried out by using a label directly or indirectly bound to the candidate compound. And easy to measure. Alternatively, the screening method may use competition with a labeled competitor. Furthermore, in such screening methods, whether a candidate compound results in a signal generated by activation or inhibition of the polypeptide can be tested using a detection system suitable for cells carrying the polypeptide. . In general, inhibitors of activation are assayed in the presence of known agonists to determine the effect of the presence of a candidate compound on agonist activation. In a screening method for an inverse agonist or inhibitor by examining whether or not a candidate compound suppresses the activation of a polypeptide in the absence of an agonist or inhibitor,
A constitutively active polypeptide is used. In addition, these screening methods consist of combining a candidate compound with a solution containing a polypeptide of the invention to form a mixture, measuring Wnt-6 activity in the mixture, and measuring the Wnt-6 activity of the mixture as a standard. It just needs to include each step to compare with. High-throughput screening assays to identify antagonists of the polypeptides of the invention can also use fusion proteins such as those made from the Fc portion and Wnt-6 polypeptides as described above (D. Bennett et al., J. Mol. Recognition, 8:52
-58 (1995) and K. Johanson et al., J. Biol. Chem., 27.
0 (16): 9459-9471 (1995)).

Further, it is possible to construct a screening method for detecting the effect of an additional compound on the intracellular production of mRNA or polypeptide using the polynucleotide, polypeptide or antibody against said polypeptide of the present invention. it can. For example, monoclonal or polyclonal antibodies can be used by standard methods known in the art to construct an ELISA assay for measuring the level of secretion or cell binding of a polypeptide, which may be performed on appropriately engineered cells or It can be used to search for substances that suppress or enhance the production of polypeptide from tissues (also called antagonists or agonists, respectively).

If a membrane-bound or soluble receptor is present, the polypeptide of the invention may be used to identify this type of receptor by standard receptor binding methods known in the art. Can be used. Such receptor binding methods include, but are not limited to, ligand binding assays and cross-linking assays in which the polypeptide is labeled with a radioactive isotope (eg, ¹²⁵ I) or chemically modified (eg. (Eg biotinylation)
Alternatively, it is fused to a peptide sequence suitable for detection and purification, and incubated with a putative receptor source (cell, cell membrane, cell supernatant, tissue extract, body fluid, etc.). Other methods include biophysical methods such as surface plasmon resonance and spectroscopy. These screening methods
It can also be used to identify agonists or antagonists of the polypeptide that compete with the binding of the polypeptide or its receptor (if present). Standard methods for conducting screening assays are well understood in the art.

Examples of potential antagonists of the polypeptides of the present invention include antibodies, in some cases oligonucleotides or proteins (eg, those in close association with the ligands, substrates, receptors, enzymes, etc. of the polypeptides). , Ligands, substrates, receptors, enzymes, etc.), or small molecules that bind the polypeptide of the invention but do not induce a response (and thus interfere with the activity of the polypeptide).

Thus, in another aspect, the invention comprises:
An agonist, an antagonist of the polypeptide of the present invention,
The present invention relates to a screening kit for identifying a compound that reduces or increases the production of a ligand, a receptor, a substrate, an enzyme, etc., or a polypeptide of this type, which kit comprises (a) the polypeptide of the present invention and (b) the present invention. A recombinant cell expressing the polypeptide of the invention, (c) a cell membrane expressing the polypeptide of the invention, or (d) an antibody against the polypeptide of the invention, said polypeptide being preferred Is the polypeptide of SEQ ID NO: 2. It will be appreciated that in such a kit, (a), (b), (c) or (d) is a substantial constituent.

Those skilled in the art will appreciate that the polypeptide of the present invention may be an agonist of the polypeptide based on its structure,
It will be readily appreciated that it can also be used in methods of designing antagonists or inhibitors. This method
(a) First, analyze the three-dimensional structure of the polypeptide, (b)
Assuming a three-dimensional structure of a likely reaction site or binding site of an agonist, antagonist or inhibitor, (c) synthesizing a candidate compound expected to bind or react with the envisioned reaction site or binding site, and
(d) determining whether the candidate compound is in fact an agonist, antagonist or inhibitor. It will be further appreciated that this is usually an iterative process.

In a further embodiment, the present invention relates to either excess or deficiency of Wnt-6 polypeptide activity, such as neurological disorders (especially schizophrenia, bipolar disorder, unipolar disorder, Alzheimer's disease and epilepsy), cancer (especially squamous cell carcinoma), cardiovascular disease, stroke, and developmental disorders
Provide treatments for abnormal conditions, including ichthyosis.

If the activity of the polypeptide is in excess, several approaches are available. One approach is to inhibit the function of the polypeptide, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc., or by reducing an abnormal condition by suppressing a second signal. An effective amount to do
It comprises administering the above inhibitor compound (antagonist) to a patient together with a pharmaceutically acceptable carrier. In another approach, soluble forms of the polypeptide that are still capable of binding the ligand, substrate, enzyme, receptor, etc. in competition with the endogenous polypeptide can be administered. A typical example of such a competitor is a fragment of the Wnt-6 polypeptide.

In yet another approach, expression inhibition methods can be used to suppress the expression of the gene encoding the endogenous Wnt-6 polypeptide. Such known techniques require the use of antisense sequences, which are either produced in the body or administered ex vivo (eg, Oligodeoxynucleotides
as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, FL (1988)
See O'Connor, J. Neurochem (1991) 56: 560). Alternatively, oligonucleotides that form triple helices with this gene can be supplied (eg, Lee et al., Nucleic Acids Res (1
979) 6: 3073; Cooney et al., Science (1988) 241: 456; Der
See van et al., Science (1991) 251: 1360). These oligomers can be administered per se or the relevant oligomers can be expressed in vivo. Synthetic antisense or triplex oligonucleotides can include modified bases or backbones. Examples of the latter include methylphosphonate, phosphorothioate or peptide nucleic acid backbones. Such scaffolds have been incorporated into antisense or triplex oligonucleotides to provide protection from nuclease degradation and are known in the art. Antisense and triplex molecules synthesized with these or other modified backbones also form part of the invention.

Furthermore, the expression of human Wnt-6 polypeptide can be blocked by using a ribozyme specific for the human Wnt-6 mRNA sequence. Ribozymes are catalytically active RNAs that may be naturally occurring or synthetic (eg, Usman, N et al., Curr. Opin. Struc.
t. Biol. (1996) 6 (4), 527-33). Synthetic ribozymes can be designed to specifically cleave human Wnt-6 mRNA at selected sites, which prevents translation of human Wnt-6 mRNA into a functional polypeptide. Ribozymes can also be synthesized using the natural ribose phosphate backbone and natural bases normally found in RNA molecules. It is also possible to synthesize ribozymes, such as 2'-O-methyl RNA, using non-natural backbones to provide protection from ribonuclease degradation, which ribozymes may include modified bases.

In treating abnormal conditions associated with underexpression of Wnt-6 and its activity, several approaches can also be taken. One approach involves administering to a patient a therapeutically effective amount of a compound that activates a polypeptide of the invention (ie, the agonist) together with a pharmaceutically acceptable carrier to alleviate the abnormal condition. It consists of Alternatively, gene therapy can be used to endogenously produce Wnt-6 in the patient's relevant cells. For example, the polynucleotides of the invention are genetically engineered for expression by a replication defective retroviral vector as described above. The retroviral expression construct is then isolated and introduced into packaging cells transduced with a retroviral plasmid vector containing RNA encoding the polypeptides of the invention. As a result, the packaging cells will produce infectious viral particles containing the gene of interest. in vivo cell manipulation and in
These producer cells are administered to the patient for expression of the polypeptide in vivo. For an overview of gene therapy, see Human Molecular Genetics, T Strachan and A PRe.
Chapter in ad, BIOS Scientific Publishers Ltd (1996)
er 20, Gene Therapy and other Molecular Genetic-bas
ed Therapeutic Approaches (and references therein)
checking ... Another approach is to administer a therapeutic amount of a polypeptide of the invention together with a suitable pharmaceutical carrier.

In a further aspect, the invention provides a therapeutically effective amount of a polypeptide (eg, a soluble form of the polypeptide of the invention), an agonist or antagonist peptide, or a small molecule compound in a pharmaceutically acceptable carrier or Provided is a pharmaceutical composition containing together with an excipient. Carriers of this type include, but are not limited to, saline, saline, dextrose, water, glycerol, ethanol, and combinations thereof. The present invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the inventive compositions described above. The polypeptides of the invention and the other compounds may be used alone or in combination with other compounds, eg therapeutic compounds.

The pharmaceutical composition may be adapted to the route of administration, eg the systemic or oral route of administration. Suitable forms for systemic administration are infusion, typically intravenous. Other injection routes such as subcutaneous, intramuscular or intraperitoneal can also be used. Another means of systemic administration is transmucosal and transdermal administration using penetrants such as bile salts, fusidic acids and other surfactants. Furthermore, oral administration is possible provided that the polypeptide of the present invention or other compound can be formulated as an enteric solution or capsule. These compounds may be topically administered and / or localized in the form of ointments, pastes, gels and the like.

The dosage range required depends on the choice of peptide or other compound of the invention, the route of administration, the nature of the formulation, the condition of the patient and the judgment of the practitioner. However, suitable dosages are in the range of 0.1-100 μg / kg of patient weight. Wide variations in the required dosage are to be expected in view of the wide variety of compounds available and the differing efficiencies of administration routes. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Such variation in dosage levels can be adjusted using standard empirical optimization procedures, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above. For example, cells from a patient are genetically engineered ex vivo with a polynucleotide such as DNA or RNA encoding a polypeptide, eg, using a retroviral plasmid vector. Thereafter, these cells are introduced into the patient.

Polynucleotide and polypeptide sequences provide a valuable information resource with which to identify other sequences of similar homology. This is maximally facilitated by storing such sequences in a computer readable medium and then using the stored data to search a sequence database with known search tools such as those in the GCG and LaserGene software packages. . Therefore, in a further aspect, the invention provides SEQ ID NO: 1.
There is provided a computer readable medium having stored thereon a polynucleotide comprising the sequence of and / or a polypeptide sequence encoded thereby.

The following definitions are intended to facilitate understanding of terms often used in the above description. As used herein, "antibody" includes polyclonal and monoclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, as well as Fab fragments that include the products of Fab or other immunoglobulin expression libraries.
"Isolated" means altered "by the hand of man" from the natural state. If an "isolated" composition or substance is naturally-occurring, it has been altered or separated from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in the body of a living animal is not "isolated", but a polynucleotide or polypeptide separated from its naturally occurring co-existing material is As used in the text, it is "isolated".

"Polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which is unmodified RNA or D.
It can be NA, or modified RNA or DNA. “Polynucleotide” includes, but is not limited to, single-stranded and double-stranded DNA, DNA in which single-stranded region and double-stranded region are mixed, single-stranded and double-stranded RNA,
RNA and DNA in which single-stranded region and double-stranded region are mixed
And RNA, including hybrid molecules, which may be single-stranded or, more typically, double-stranded and may be a mixture of single-stranded and double-stranded regions. in addition,
"Polynucleotide" means RNA or DNA or RN
It means a triple-stranded region consisting of both A and DNA. The term "polynucleotide" also includes DNAs or RNAs that contain one or more modified bases, and DNAs or RNAs that have a backbone that has been modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA. Thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms of polynucleotides commonly found in nature, as well as chemical forms of DNA and RNA characteristic of viruses and cells. . "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

By "polypeptide" is meant a peptide or protein containing two or more amino acids linked by peptide bonds or modified peptide bonds (ie, peptide isosteres). "Polypeptide"
Refers to both short chains (commonly referred to as peptides, oligopeptides or oligomers) and long chains (commonly referred to as proteins). Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. A "polypeptide" comprises an amino acid sequence modified either by natural processes, such as post-translational processing, or by chemical modification methods known in the art. Such modifications are detailed in basic textbooks, more detailed academic and research literature. Modifications can be made anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. The polypeptide may be branched for ubiquitination, or cyclic with or without branching. Cyclic, branched, or branched cyclic polypeptides may result from post-translational natural processes or may be produced synthetically. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, flavin covalent attachment,
Covalent bond of heme moiety, covalent bond of nucleotide or nucleotide derivative, covalent bond of lipid or lipid derivative,
Phosphatidylinositol covalent bond, crosslinking, cyclization,
Disulfide bond formation, demethylation, covalent cross-link formation, cystine formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation , Oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as arginylation, ubiquitination, etc. (eg,
Proteins-Structure and Molecular Properties, 2nd
Ed., TE Creighton, WH Freeman and Company, Ne
w York, 1993; Posttranslational Covalent Modificat
ion of Proteins, BC Johnson, Academic Press, N
Wold, F., Post-translational Prote in ew York, 1983
in Modifications: Perspectives and Prospects, 1-12
Page; Seifter et al., “Analysis for protein modification
sand nonprotein cofactors ", Meth Enzymol (1990) 18
2: 626-646; and Rattan et al., “Protein Synthesis: Po.
st-translational Modifications and Aging ", Ann NY
See Acad Sci (1992) 663: 48-62).

As used herein, a "variant" is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains essential properties. Typical polynucleotide variants differ in nucleotide sequence from the reference polynucleotide. Changes in the nucleotide sequence of this variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, deletions, additions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. Typical polypeptide variants differ in amino acid sequence from a reference polypeptide. Generally, the sequence of the reference polypeptide and the sequence of the variant are generally similar and limited to differences that are identical in many regions. Any combination of variant and reference polypeptide 1
The amino acid sequences may differ due to the above substitutions, deletions and additions. The amino acid residue that is replaced or inserted may or may not be one encoded by the genetic code. Variants of polynucleotides or polypeptides may be naturally occurring variants, such as allelic variants, or variants not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis or direct synthesis. "Identity," as known in the art, is a relationship between two or more such sequences, as determined by comparing the polypeptide or polynucleotide sequences. In the art, "identity" also refers to the match between strands of a polypeptide or polynucleotide sequence.
It means the degree of sequence relatedness between such sequences, as determined by (match). “Identity” and “similarity” can be calculated without difficulty by known methods, and as such a method, for example, Computational Molecular Biolog
y, Lesk, AM, Oxford University Press, New Yor
k, 1988; Biocomputing: Informatics and Genome Proje
cts, Smith, DW Edition, Academic Press, New York, 199
3; Computer Analysis of Sequence Data, Part I, Gri
ffin, AM and Griffin, HG, Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biolo
gy, von Heinje, G., Academic Press, 1987; Sequence
Analysis Primer, Gribskov, M. and Devereux, J.
Edited by M Stockton Press, New York, 1991; and Carill.
o, H. and Lipman, D., SIAM J. Applied Math., 48: 1
073 (1988), but not limited thereto. Preferred methods to determine identity are designed to give the largest match between the sequences under consideration.
Moreover, methods to determine identity and similarity are codified in publicly available computer programs. Two
Suitable computer program methods for determining identity and similarity between sequences include the GCG program package (Devereux, J. et al., Nucleic Acids Research 12 (1): 3.
87 (1984)), BLASTP, BLASTN and FAS
TA (Atschul, SF et al., J. Molec. Biol. 215: 403-410.
(1990)), but is not limited to these. BLASTX
The program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM
NIH Bethesda, MD 20894; Altschul, S. et al., J. Mol. Bi
ol. 215: 403-410 (1990)). The well known Smith Waterman algorithm can also be used to determine identity.

Suitable parameters for comparing polypeptide sequences include the following: 1) Algorithm: Needleman & Wunsch, J. Mol. Bio.
l. 48: 443-453 (1970); Comparison matrix: BLOSSUM62, Hentikoff and Hentik
off, Proc. Natl. Acad. Sci. USA, 89: 10915-10919
(1992) Gap Penalty: 12 Gap Length Penalty: 4 A useful program with these parameters is Gen
It is publicly available as the "gap" program from the etics Computer Group (Madison WI). The above parameters are the default parameters (def
ault parameter) (there is no end gap penalty).

Suitable parameters for comparing polynucleotide sequences include the following: 1) Algorithm: Needleman & Wunsch, J. Mol. Bio.
l. 48: 443-453 (1970); Comparison Matrix: Match = +10, Mismatch = 0 Gap penalty: 50 Gap length penalty: 3 A useful program with these parameters is Gen.
It is available as the "gap" program from the etics Computer Group (Madison WI). The above parameters are the default parameters for polynucleotide comparison.

By way of example, the polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: 1, ie 100% identical, or may contain up to an integer number of nucleotide variations relative to the reference sequence. Good. The mutations are selected from the group consisting of deletions, substitutions (including transitions and transversions) or additions of at least one nucleotide, such mutations of the reference nucleotide sequence.
It may occur at the 5'or 3'terminal positions, or anywhere between these terminal positions, intervening individually between the nucleotides in the reference sequence or as one or more contiguous groups within the reference sequence. it can. The number of nucleotide variations is determined by multiplying the total number of nucleotides of SEQ ID NO: 1 by the respective% identity (divided by 100) value and subtracting the product from the total number of nucleotides of SEQ ID NO: 1, ie: It can be obtained by n _n ≦ x _n − (x _n · y). Where n _n is the number of nucleotide mutations, x _n is the total number of nucleotides of SEQ ID NO: 1, y is, for example, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 90% for 85% Is 0.90,
For 95%, it is 0.95, etc. The product of non-integers of x _n and y is rounded down to the nearest integer before subtracting the product from x _n . Modification of the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 results in nonsense, missense or frameshift mutations in the coding sequence, thereby altering the polypeptide encoded by the polynucleotide after such mutations. be able to.

Similarly, the polypeptide sequence of the present invention is identical to the reference sequence of SEQ ID NO: 2, ie, even if it has 100% identity, it contains up to an integer number of amino acid mutations relative to the reference sequence. The percent identity may be less than 100%.
Said mutation is selected from the group consisting of a deletion, substitution (including conservative and non-conservative amino acid substitutions) or addition of at least one amino acid, such mutation being the amino or carboxy terminal position of the reference polypeptide sequence, or these termini. It may be located between any of the positions and may be interspersed between amino acids in the reference sequence individually or as one or more contiguous groups within the reference sequence. To determine the number of amino acid mutations for a given% identity, multiply the total number of amino acids in SEQ ID NO: 2 by their respective% identity values (divided by 100) and subtract the product from the total number of amino acids in SEQ ID NO: 2. by, i.e., the following equation: n _a ≦ x _a - can be obtained by (x _a · y). In the formula, n _a is the number of amino acid mutations, x _a is the total number of amino acids in SEQ ID NO: 2, and y is 0.70 for 70% and 0 for 80%, for example.
It is 0.85 for 80% and 85%, and the product of non-integers of x _a and y is rounded down to the nearest integer before subtracting the product from x _a .

“Homolog” is a general term used in the art to indicate a polynucleotide or polypeptide having a high degree of sequence relatedness to a subject sequence. Such relatedness can be quantified by determining the degree of identity and / or similarity between the sequences to be compared, as described above. "Ortholog" means a polynucleotide or polypeptide that is a functional equivalent of the polynucleotide or polypeptide in another species.
Included within this general term is the term (ortholog), and the term "paralog", which means sequences that are functionally similar when considered within the same species.

A "fusion protein" is a protein encoded by two, often unrelated, fused genes or fragments thereof. As an example, EP-A
-0 464 describes fusion proteins comprising various parts of the constant region of immunoglobulin molecules and other human proteins or parts thereof. In many cases, for therapeutic and diagnostic use, it will be advantageous to use the immunoglobulin Fc region as part of a fusion protein, which may, for example, improve pharmacokinetic properties (eg EP-A-0232). See 262). On the other hand, for some uses it may be desirable to remove the Fc portion after expressing, detecting and purifying the fusion protein.

All publications, including patents and patent application publications, cited herein are incorporated by reference in their entirety as if each publication was explicitly and individually indicated. Incorporated here.

[0069] sequence information SEQ ID NO: 1 GGCACGAGCGCAGGAGACACAGGCGCTGGCTGCCCCGTCCGCTCTCCGCCTCCGCCGCGCCCTCCTCGCCCG GGATGGGCCCCCCCGCCGCCGCCGGATCCCTCGCCTCCCGGCCGCCGCCGTTGCGCTCGCCGCGCTCGCACT GAAGCCCGGGCCCTCGCGCGCCGCGGTTCGCCCCGCAGCCTCGCCCCCTGCCCACCCGGGCGGCCGTAGGGC GGTCACGATGCTGCCGCCCTTACCCTCCCGCCTCGGGCTGCTGCTGCTGCTGCTCCTGTGCCCGGCGCACGT CGGCGGACTGTGGTGGGCTGTGGGCAGCCCCTTGGTTATGGACCCTACCAGCATCTGCAGGAAGGCACGGCG GCTGGCCGGGCGGCAGGCCGAGTTGTGCCAGGCTGAGCCGGAAGTGGTGGCAGAGCTAGCTCGGGGCGCCCG GCTCGGGGTGCGAGAGTGCCAGTTCCAGTTCCGCTTCCGCCGCTGGAATTGCTCCAGCCACAGCAAGGCCTT TGGACGCATCCTGCAACAGGACATTCGGGAGACGGCCTTCGTGTTCGCCATCACTGCGGCCGGCGCCAGCCA CGCCGTCACGCAGGCCTGTTCTATGGGCGAGCTGCTGCAGTGCGGCTGCCAGGCGCCCCGCGGGCGGGCCCC TCCCCGGCCCTCCGGCCTGCCCGGCACCCCCGGACCCCCTGGCCCCGCGGGCTCCCCGGAAGGCAGCGCCGC CTGGGAGTGGGGAGGCTGCGGCGACGACGTGGACTTCGGGGACGAGAAGTCGAGGCTCTTTATGGACGCGCG GCACAAGCGGGGACGCGGAGACATCCGCGCGTTGGTGCAACTGCACAACAACGAGGCGGGCAGGCTGGCCGT GCGGAGCCACACGCGCACCGAGTGCAAATGCCACGGGCTGTCGGGATCATGCGCGCTGCGCACCTGCTGGCA GAAG CTGCCTCCATTTCGCGAGGTGGGCGCGCGGCTGCTGGAGCGCTTCCACGGCGCCTCACGCGTCATGGG CACCAACGACGGCAAGGCCCTGCTGCCCGCCGTCCGCACGCTCAAGCCGCCGGGCCGAGCGGACCTCCTCTA CGCCGCCGATTCGCCCGACTTTTGCGCCCCCAACCGACGCACCGGCTCCCCCGGCACGCGCGGTCGCGCCTG CAATAGCAGCGCCCCGGACCTCAGCGGCTGCGACCTGCTGTGCTGCGGCCGCGGGCACCGCCAGGAGAGCGT GCAGCTCGAAGAGAACTGCCTGTGCCGCTTCCACTGGTGCTGCGTAGTACAGTGCCACCGTTGCCGTGTGCG CAAGGAGCTCAGCCTCTGCCTGTGACCCGCCGCCCGGCCGCTAGACTGACTTCGCGCAGCGGTGGCTCGCAC CTGTGGGACCTCAGGGCACCGGCACCGGGCGCCTCTCGCCGCTCGAGCCCAGCCTCTCCCTGCCAAAGCCCA ACTCCCAGGGCTCTGGAAATGGTGAGGCGAGGGGCTTGAGAGGAACGCCCACCCACGAAGGCCCAGGGCGCC AGACGGCCCCGAAAAGGCGCTCGGGGAGCGTTTAAAGGACACTGTACAGGCCCTCCCTCCCCTTGGCCTCTA GGAGGAAACAGTTTTTTAGACTGGAAAAAAGCCAGTCTAAAGGCCTCTGGATACTGGGCTCCCCAGAACTGC TGGCCACAGGATGGTGGGTGAGGTTAGTATCAATAAAGATATTTAA SEQ ID NO: 2 MLPPLPSRLGLLLLLLLCPAHVGGLWWAVGSPLVMDPTSICRKARRLAGRQAELCQAEPEVVAELARGARLG VRECQFQFRFRRWNCSSHSKAFGRILQQDIRETAFVFAITAAGASHAVTQACSMGELLQCGCQAPRGRAPPR PSGLPGTPGPPGPAGSPEGSAAWEWGGCGDDVDFGDEKSRLFMDARHKRGRGDIRALVQLHNNEA GRLAVRS HTRTECKCHGLSGSCALRTCWQKLPPFREVGARLLERFHGASRVMGTNDGKALLPAVRTLKPPGRADLLYAA DSPDFCAPNRRTGSPGTRGRACNSSAPDLSGCDLLCCGRGHRQESVQLEENCLCRFHWCCVVQCHRCRVRKE LSLCL

[0070]

[Sequence list]                             SEQUENCE LISTING    <110> SmithKline Beecham plc    <120> Wnt-6 Polypeptides and Wnt-6 Polynucleotides    <130> PA98-385    <150> U.K. Application No. 9817586.2 <151> 1998-8-12    <160> 2    <170> FastSEQ for Windows Version 3.0    <210> 1 <211> 1702 <212> DNA <213> Homo sapiens    <400> 1 ggcacgagcg caggagacac aggcgctggc tgccccgtcc gctctccgcc tccgccgcgc 60    cctcctcgcc cgggatgggc ccccccgccg ccgccggatc cctcgcctcc cggccgccgc 120    cgttgcgctc gccgcgctcg cactgaagcc cgggccctcg cgcgccgcgg ttcgccccgc 180    agcctcgccc cctgcccacc cgggcggccg tagggcggtc acgatgctgc cgcccttacc 240    ctcccgcctc gggctgctgc tgctgctgct cctgtgcccg gcgcacgtcg gcggactgtg 300    gtgggctgtg ggcagcccct tggttatgga ccctaccagc atctgcagga aggcacggcg 360    gctggccggg cggcaggccg agttgtgcca ggctgagccg gaagtggtgg cagagctagc 420    tcggggcgcc cggctcgggg tgcgagagtg ccagttccag ttccgcttcc gccgctggaa 480    ttgctccagc cacagcaagg cctttggacg catcctgcaa caggacattc gggagacggc 540    cttcgtgttc gccatcactg cggccggcgc cagccacgcc gtcacgcagg cctgttctat 600    gggcgagctg ctgcagtgcg gctgccaggc gccccgcggg cgggcccctc cccggccctc 660    cggcctgccc ggcacccccg gaccccctgg ccccgcgggc tccccggaag gcagcgccgc 720    ctgggagtgg ggaggctgcg gcgacgacgt ggacttcggg gacgagaagt cgaggctctt 780    tatggacgcg cggcacaagc ggggacgcgg agacatccgc gcgttggtgc aactgcacaa 840    caacgaggcg ggcaggctgg ccgtgcggag ccacacgcgc accgagtgca aatgccacgg 900    gctgtcggga tcatgcgcgc tgcgcacctg ctggcagaag ctgcctccat ttcgcgaggt 960    gggcgcgcgg ctgctggagc gcttccacgg cgcctcacgc gtcatgggca ccaacgacgg 1020    caaggccctg ctgcccgccg tccgcacgct caagccgccg ggccgagcgg acctcctcta 1080    cgccgccgat tcgcccgact tttgcgcccc caaccgacgc accggctccc ccggcacgcg 1140    cggtcgcgcc tgcaatagca gcgccccgga cctcagcggc tgcgacctgc tgtgctgcgg 1200    ccgcgggcac cgccaggaga gcgtgcagct cgaagagaac tgcctgtgcc gcttccactg 1260    gtgctgcgta gtacagtgcc accgttgccg tgtgcgcaag gagctcagcc tctgcctgtg 1320    acccgccgcc cggccgctag actgacttcg cgcagcggtg gctcgcacct gtgggacctc 1380    agggcaccgg caccgggcgc ctctcgccgc tcgagcccag cctctccctg ccaaagccca 1440    actcccaggg ctctggaaat ggtgaggcga ggggcttgag aggaacgccc acccacgaag 1500    gcccagggcg ccagacggcc ccgaaaaggc gctcggggag cgtttaaagg acactgtaca 1560    ggccctccct ccccttggcc tctaggagga aacagttttt tagactggaa aaaagccagt 1620    ctaaaggcct ctggatactg ggctccccag aactgctggc cacaggatgg tgggtgaggt 1680    tagtatcaat aaagatattt aa 1702    <210> 2 <211> 365 <212> PRT <213> Homo sapiens <400> 2  Met Leu Pro Pro Leu Pro Ser Arg Leu Gly Leu Leu Leu Leu Leu Leu   1 5 10 15  Leu Cys Pro Ala His Val Gly Gly Leu Trp Trp Ala Val Gly Ser Pro              20 25 30  Leu Val Met Asp Pro Thr Ser Ile Cys Arg Lys Ala Arg Arg Leu Ala          35 40 45  Gly Arg Gln Ala Glu Leu Cys Gln Ala Glu Pro Glu Val Val Ala Glu      50 55 60  Leu Ala Arg Gly Ala Arg Leu Gly Val Arg Glu Cys Gln Phe Gln Phe  65 70 75 80  Arg Phe Arg Arg Trp Asn Cys Ser Ser His Ser Lys Ala Phe Gly Arg                  85 90 95  Ile Leu Gln Gln Asp Ile Arg Glu Thr Ala Phe Val Phe Ala Ile Thr              100 105 110  Ala Ala Gly Ala Ser His Ala Val Thr Gln Ala Cys Ser Met Gly Glu          115 120 125  Leu Leu Gln Cys Gly Cys Gln Ala Pro Arg Gly Arg Ala Pro Pro Arg      130 135 140  Pro Ser Gly Leu Pro Gly Thr Pro Gly Pro Pro Gly Pro Ala Gly Ser  145 150 155 160  Pro Glu Gly Ser Ala Ala Trp Glu Trp Gly Gly Cys Gly Asp Asp Val                  165 170 175  Asp Phe Gly Asp Glu Lys Ser Arg Leu Phe Met Asp Ala Arg His Lys              180 185 190  Arg Gly Arg Gly Asp Ile Arg Ala Leu Val Gln Leu His Asn Asn Glu          195 200 205  Ala Gly Arg Leu Ala Val Arg Ser His Thr Arg Thr Glu Cys Lys Cys      210 215 220  His Gly Leu Ser Gly Ser Cys Ala Leu Arg Thr Cys Trp Gln Lys Leu  225 230 235 240  Pro Pro Phe Arg Glu Val Gly Ala Arg Leu Leu Glu Arg Phe His Gly                  245 250 255  Ala Ser Arg Val Met Gly Thr Asn Asp Gly Lys Ala Leu Leu Pro Ala              260 265 270  Val Arg Thr Leu Lys Pro Pro Gly Arg Ala Asp Leu Leu Tyr Ala Ala          275 280 285  Asp Ser Pro Asp Phe Cys Ala Pro Asn Arg Arg Thr Gly Ser Pro Gly      290 295 300  Thr Arg Gly Arg Ala Cys Asn Ser Ser Ala Pro Asp Leu Ser Gly Cys  305 310 315 320  Asp Leu Leu Cys Cys Gly Arg Gly His Arg Gln Glu Ser Val Gln Leu                  325 330 335  Glu Glu Asn Cys Leu Cys Arg Phe His Trp Cys Cys Val Val Gln Cys              340 345 350  His Arg Cys Arg Val Arg Lys Glu Leu Ser Leu Cys Leu          355 360 365

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 39/395 A61K 45/00 45/00 48/00 48/00 C07K 14/47 C07K 14/47 16/18 16 / 18 C12N 1/10 101 C12N 1/10 101 1/15 1/15 1/19 1/19 1/21 1/21 C12P 21/02 C 5/10 G01N 33/53 D C12P 21/02 33/577 B G01N 33/53 C12P 21/08 33/577 A61K 37/02 // C12P 21/08 C12N 5/00 A (72) Inventor Michael Earl Barnes England UK 195 AD Essex, Harlow, The Pinnacles ， Cold Harbor Road Smith Klein Beechham Pharmaceuticals (72) Inventor Tania Tamson Tester UK UK 195 Eddie Essex, Harlow, The Pinnacles, Cold Harbor Road Smith Kline Beechham Pharmaceuticals Tikaruzu

Claims (20)

[Claims] 1. An isolated polypeptide comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. 2. The polypeptide of claim 1, wherein the amino acid sequences have at least 99% identity. 3. The polypeptide according to claim 1, comprising the amino acid sequence of SEQ ID NO: 2. 4. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. 5. An isolated polynucleotide comprising a nucleotide sequence that encodes a polypeptide having at least 98% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2, or the polynucleotide. A polynucleotide consisting of a nucleotide sequence complementary to. 6. An isolated polynucleotide comprising a nucleotide sequence having at least 90% identity to a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, or complementary to said polynucleotide. A polynucleotide consisting of a specific nucleotide sequence. 7. An isolated polynucleotide comprising a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1, or a nucleotide complementary to the polynucleotide. A polynucleotide consisting of a sequence. 8. The polynucleotide according to either claim 6 or 7, wherein said identity is at least 95%. 9. An isolated polynucleotide selected from the following (a) to (c), or a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide: (a) from the amino acid sequence of SEQ ID NO: 2 A polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising: (b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1; and (c) an appropriate library under the stringent hybridization conditions of SEQ ID NO: A polynucleotide obtained by screening using a labeled probe having the nucleotide sequence of 1 or a fragment thereof. 10. An expression system containing a polynucleotide capable of producing the polypeptide of claim 1 when the expression system described below is present in a compatible host cell. 11. A host cell containing the expression system of claim 10, or a membrane thereof expressing the polypeptide of claim 1. 12. Culturing the host cell of claim 11 under conditions sufficient to produce the polypeptide of claim 1 and recovering the polypeptide from the culture medium. A method for producing the polypeptide according to claim 1. 13. An antibody immunospecific for the polypeptide of claim 1. 14. A screening method for identifying a compound that stimulates or suppresses the function of the polypeptide according to claim 1, which comprises (a) a candidate compound and the polypeptide (or a protein carrying the polypeptide). Cell or its membrane) or its fusion protein is measured by a label directly or indirectly bound to the candidate compound, (b) the candidate compound and the polypeptide (or the polypeptide) Binding to cells or their membranes) or their fusion proteins, in the presence of a labeled competitor, (c) the candidate compound results in a signal generated by activation or inhibition of the polypeptide. The use of a detection system suitable for cells or cell membranes carrying the polypeptide, (d) the candidate compound, and the method according to claim 1. Preparing a mixture with a solution containing the polypeptide and measuring the activity of the polypeptide in the mixture and comparing the activity of the mixture with a standard, and (e) the candidate compound A screening method comprising a method selected from the group consisting of detecting the mRNA encoding the polypeptide and the effect on the production of the polypeptide. 15. An agonist or antagonist to the polypeptide according to any one of claims 1 to 4. 16. A compound selected from the following (a) to (c) for use in therapy: (a) an agonist or antagonist to the polypeptide according to any one of claims 1 to 4, (b) ) A nucleic acid molecule which modulates the expression of the polynucleotide according to any one of claims 5 to 9 and (c) the nucleotide sequence encoding the polypeptide according to claim 1. 17. A method for examining a disease associated with expression or activity of the polypeptide of claim 1 in a subject or susceptibility to the disease, comprising: (a) Determining whether the nucleotide sequence encoding the peptide has a mutation, and / or (b) analyzing the presence or amount of expression of said polypeptide in a sample obtained from said subject. . 18. An isolated polypeptide of (a) or (b) below: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2,
Or (b) a polypeptide having an amino acid sequence of SEQ ID NO: 2 in which at least one amino acid has been deleted, substituted or added, and having Wnt-6 activity. 19. A polynucleotide encoding the polypeptide according to claim 18. 20. An isolated polynucleotide of (a) or (b) below: (a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, or (b) a polynucleotide which is stringent with the polynucleotide of (a). A polynucleotide encoding a polypeptide which hybridizes under conditions and has Wnt-6 activity.